Refractive index matching formulations

ABSTRACT

Refractive index matching (RIM) formulations, their use for mounting biological specimens (e.g., cells and tissues) to a substrate, and methods of visualizing biological specimens embedded in RIM formulations are described.

TECHNICAL FIELD

Refractive index matching (RIM) formulations, their use for mountingbiological specimens (e.g., cells and tissues) to a substrate, andmethods of visualizing biological specimens embedded in RIM formulationsare described.

BACKGROUND

Biological specimens often are mounted on an optically clear substrate,such as a glass microscope slide, for examination by light microscopy.Typically, the mounting process involves suspending a biologicalspecimen (e.g., a fixed cell or tissue) in a mounting solution, alsoreferred to as a “mounting medium” or “mountant,” and then depositingthe specimen onto the surface of the substrate, thereby embedding thespecimen in the mounting solution. The mounting solution and embeddedspecimen are optionally dried prior to interrogation, typically under acover slide. The mounting medium protects the specimen from physicaldamage and allows for extended storage of the specimen.

The selection of a mounting medium is dictated by the type of sample andsubstrate. The mounting medium can be liquid or can harden into apermanent mountant. In addition, the mounting media ideally does notreact with the specimen and does not crystallize or darken over time. Ifthe specimen is stained with a dye, the mountant also should not causethe dye to fade or bleach. For biological specimens that are labeledwith fluorescent dyes, selection of a suitable mountant often isgoverned by its ability to minimize photobleaching A suitable mountantshould effectively reduce photobleaching of the fluorophores, whileminimizing quenching of the initial fluorescence intensity. Loss offluorescence through irreversible photobleaching can lead to asignificant reduction in sensitivity, particularly when target moleculesare of low abundance or when excitation light is of high intensity orlong duration. Another factor in the selection of an appropriatemountant is its optical clarity. Optical clarity is influenced not justby how clear a mountant is, but by how well the refractive index (RI) ofthe mountant, sample, and glass or other substrate are matched. Forbiological specimens, an aqueous mounting medium is generally chosen.However, commercially available, aqueous mounting media fail toadequately match the refractive index of the specimen. For example,cells and tissue typically have a RI of 1.35-1.42. Because the RI ofthese mounting media fall below the RI of the glass used in microscopeslides and coverslips (1.50-1.54) and also do not match the RI of thebiological specimens, these mounting media do not permit for optimaloptical clarity. Thus, there is a need for improved formulations formounting biological specimens that have a RI that more closely matchesthe RI of glass and/or the biological specimen under interrogation.

SUMMARY

Refractive index matching (RIM) solutions are provided that include awater-soluble polymer, wherein the polymer has a molar refraction(R_(LL)) to molar volume (V_(m)) ratio of 0.27 to 0.34, and a polyol. Inone aspect, a refractive index matching (RIM) solution is provided thatincludes a water-soluble polymer, wherein the polymer has a molarrefraction (R_(LL)) to molar volume (V_(m)) ratio of 0.27 to 0.34; and apolyol, wherein the weight ratio of the water-soluble polymer to thepolyol is 0.125:1 to 4:1. In certain embodiments, the weight ratio ofwater-soluble polymer to polyol is 0.318:1 to 4:1. The RIM solution canfurther include water or a buffer. The RIM solution can have arefractive index (RI) of 1.33 or greater (e.g., 1.333 to 1.530; or 1.330to 1.420). In certain embodiments, the RI of the RIM solution is 1.36 orgreater. In certain embodiments, the RI of the RIM solution is 1.38 orgreater. In yet other embodiments, the RI of the RIM solution is 1.47 orgreater. Typically, the RI of the RIM solution is 1.60 or less.

In another aspect, a method of mounting a biological specimen on asubstrate is provided that includes depositing the biological specimenon the substrate; and contacting the biological specimen with the RIMsolution disclosed herein to provide a mounted sample. In any of themethods, mounted biological specimens or kits described herein, thesubstrate can be a microscope slide, cuvette, well, imaging chamber ordish. Representative examples of biological specimens include a cell,tissue, 3D cell culture (e.g., spheroid/organoid), or a whole organism(e.g. fruit fly, worm, zebra fish). Optionally, the biological specimenis labeled with a fluorescent dye or fluorescent protein. The method canfurther include drying the mounted sample, whereby the water-solublepolymer solidifies to provide a solidified polymer. The water-solublepolymer can solidify in about 48 hours or less; or 24 hours or less atroom temperature; or in about 4 hours or less at a temperature of about40° C. The RI of the solidified polymer can exceed 1.47. In certainembodiments, the RI of the solidified polymer is 1.48 to 1.54. Inspecific embodiments, the RI of the solidified polymer is 1.50 to 1.525.The method can further include visualizing the biological specimen onthe substrate with a microscope.

In yet another aspect, a solidified mountant is provided that includes awater-soluble polymer, wherein the polymer has a molar refraction(R_(LL)) to molar volume (V_(m)) ratio of 0.27 to 0.34, and a polyol,wherein the weight ratio of the water-soluble polymer to the polyol is0.125:1 to 4:1. The weight ratio of the water-soluble polymer to thepolyol in the solidified mountant can be 0.5:1 or greater. Thesolidified mountant can have a refractive index of 1.47 or greater. Insome embodiments, the refractive index of the solidified mountant isgreater than 1.47. In certain embodiments, a solidified mountant isprovided that includes a water-soluble polymer, wherein the polymer hasa molar refraction (R_(LL)) to molar volume (V_(m)) ratio of 0.27 to0.34, and a polyol, wherein the weight ratio of the water-solublepolymer to the polyol is 0.318:1 to 4:1, and wherein the solidifiedmountant has a refractive index of 1.48 or greater. The RI of the RIMsolutions or solidified mountants disclosed herein are ideally suited tomatch the refractive index of soda-lime glass, borosilicate glass, orimmersion oil (e.g., 1.48-1.50; or 1.50 to 1.52; or 1.52 to 1.54).

The water-soluble polymer used in the disclosed formulations can beuncharged. For example, the uncharged, water-soluble polymer can be apoly(acrylamide), poly(methacrylamide), poly(methyl vinyl ether),poly(vinyl pyrrolidone) (PVP), polyvinyl alcohol (PVA),poly(-ethyl-2-oxazoline), or poly(-methyl-2-oxazoline). In certainembodiments, the uncharged, water-soluble polymer includes a monomerresidue of N—R acrylamide or N—R methacrylamide, wherein R is methyl,ethyl, propyl, isopropyl or H; N,N-dimethylacrylamide,N,N-dimethylmethacrylamide, N,N-diethylacrylamide, N, Ndiethylmethacrylamide; N-2-hydroxypropyl methacrylamide); or acombination thereof. In certain embodiments, the uncharged,water-soluble polymer is poly(N-methyl methacrylamide) (PMMAm).Alternatively, the water-soluble polymer used in the disclosedformulations can be charged. Representative charged, water-solublepolymers include, e.g., polyacrylic acid, polymethacrylic acid,poly(diallyldimethylammonium chloride), poly(sodium-4-styrenesulfonate),poly(ethyleneimine), poly(N,N-dimethylaminoethyl acrylate),poly(N,N-diethylethylamino acrylate), poly(allylamine),poly[bis(2-chloroethyl)ether-co-1,3-bis[3-(dimethylamino)propyl]urea];or poly(vinylsulfonic acid, sodium salt). In certain embodiments, thewater-soluble polymer is not a polyol. The water-soluble polymer canhave a weight average molecular weight of about 1 kDa to about 100 kDa.In certain embodiments the weight average molecular weight is about 1kDa to about 20 kDa; or about 20 kDa to about 100 kDa; or about 48 kDato about 80 kDa. The water-soluble polymer also can be a copolymerformed from two or more of the charged or uncharged monomers describedherein, where the copolymer can be, e.g., a random, gradient, block orgraft copolymer.

The mounting formulations disclosed herein also can include a polyolsuch as polyvinyl alcohol, glycerol, diglycerol, polyglycerol, mannitol,sorbitol, erythritol, thiodiethanol, thiodipropanol, or a combinationthereof. Optionally, the formulations can further include ananti-oxidant (e.g., about 1% by weight or less), such as, e.g.,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 3-carboxyproxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate,ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine,1,4-diazabicyclo[2.2.2]octane, or a combination thereof.

In yet another aspect, a mounted biological specimen is provided thatincludes a biological specimen disposed on a substrate, wherein thebiological specimen is embedded in a solidified mountant, as disclosedherein.

In yet another aspect, a kit for mounting a biological specimen on asubstrate is provided that includes a refractive index matching (RIM)solution, as disclosed herein; and instructions for mounting thebiological specimen on the substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the refractive index change as a function oftime for Sample 8 and 13.

FIG. 2. is a plot showing axial resolution as a function of focal depthfor Sample 8, 10 and 13.

FIG. 3. is a plot showing lateral resolution as a function of focaldepth for Sample 8, 10 and 13.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, the term “about”, when used to describe a numericalvalue, encompasses a range up to ±15% of that numerical value, unlessthe context clearly dictates otherwise.

While compositions and methods are described in terms of “comprising”various components or steps (interpreted as meaning “including, but notlimited to”), the compositions and methods can also “consist essentiallyof” or “consist of” the various components and steps, such terminologyshould be interpreted as defining essentially closed-member groups.

“Refractive index” or “RI,” as used herein, is a measure of how fastlight travels through a particular medium. When light travels betweentwo media with different RI values, such as air and water, the path thatit travels is bent, distorting the image. Refractive index also is ameasure of how much the speed and the wavelength of radiation arereduced with respect to the wavelength of the light in a vacuum. BecauseRI is a ratio of two velocities, it is dimensionless.

“Water-soluble” is used herein to mean the compound can be soluble ordispersible in an aqueous-based solution, such as in water orwater-based solutions or buffer solutions, including those used inbiological or molecular detection systems as known by those skilled inthe art. A compound is considered water-soluble if it can be dissolvedin an aqueous formulation at a concentration of 10 mg/mL or more.Compounds that can be dissolved in an aqueous system at a concentrationof 100 mg/mL or greater are considered highly water-soluble. Compoundsthat can be dissolved in an aqueous system at a concentration of 1 mg/mLor less are considered to have poor water solubility.

“Biological specimen” or “biological sample,” as used herein encompasseshematological, cytological and histological specimens, such as cells, 3Dcell cultures (e.g. spheroids and organoids), tissues, whole organisms(e.g. flies, worms, zebrafish), cell-free extracts, or a fluid sample(e.g., blood or sputum). A tissue specimen can be any type of nervous,epithelial, muscular, and connective tissue, including an organ tissue.Biological samples can be from a plant or animal (e.g., human, mouse,fly, worm, fish, frog, fungi, and the like).

Formulations for use in mounting a specimen on a substrate are describedherein. The described formulations are aqueous-based systems thatprovide a hard-setting mounting medium for protection and storage of thespecimen. The formulations can be applied directly to cells and tissuesamples, which optionally can be fluorescently labeled, on a substrateand can harden upon drying. Formulations can harden without a coverslip,thus eliminating the requirement that a cover slip be used to protectthe mounted specimen. The mounting formulations disclosed herein holdthe specimen in place on the substrate, thereby stabilizing andpreserving the biological specimen on the substrate for subsequentinterrogation. For example, once mounted, the biological specimen can beimaged under a microscope. The disclosed formulations have favorableoptical characteristics that make these formulations ideal for use as amounting medium for biological samples. In particular, the disclosedformulations exhibit a high refractive index. It is known that the RI ofthe specimen and mounting medium can significantly impact image quality.For example, the difference between the RI of the specimen and thesurrounding mountant can influence how the specimen appears under amicroscope. If the difference between the RI of the specimen and thesurrounding medium is large, strong refraction of light at the specimenand mounting medium interface can occur. A large difference in RI cancause artifacts as light is refracted that can obscure details of thespecimen. In contrast, small differences in RI between the specimen andthe mounting medium can reduce light refraction, making many types ofspecimens appear brighter and/or more transparent under the microscope.

Provided herein are formulations that can match the refractive index ofa biological sample, as well as substrates, lens, coverslips, and othercomponents commonly used in microscopy. Thus, the formulations describedherein have a refractive index (RI) that matches the RI of the glass ofthe objective lens of the microscope, as well as the glass of themicroscope coverslip or other components commonly used in microscopicimaging of samples such as immersion oil. By virtue of having high RI,the disclosed formulations minimize distortion of microscope imageresulting from light refraction, making these formulations suitable foruse under high magnification and/or with immersion oils, such as aretypically used to minimize distortion of the microscope image. Oncedried, the instant formulations have an RI that exceeds the RI ofexisting hard mount formulations, while reducing photobleaching of thesample and dyes, if present. For example, particular formulationsdescribed herein can have an RI of 1.47 or greater, which issignificantly higher than standard hard-mount formulations known tothose skilled in the art.

The formulations provided herein are optically clear, even once dried,are chemically compatible with the biological specimen and do not harmor degrade the biological specimen, even after prolonged storage. Thedescribed formulations also improve clarity of higher magnification and3D reconstruction (Z-stack) images and can reduce spherical aberrationor scattered light, thereby allowing for capture of clearer images atmultiple depths. Due to the favorable physical and optical properties,the instant formulations can be implemented in three-dimensional imagingof various types of biological specimens (e.g., tissues and cells) usinga range of microscope techniques such as those used for visualization offluorophores at depth within a specimen (e.g., confocal fluorescencemicroscopy). Further, the disclosed formulations also exhibit low bubbleformation, less shrinking than other commercially available hard setmountants upon drying and do not crack upon drying and/or freezing. Themountant does not discolor or shrink when cured, making it possible totake high quality images weeks or even months after mounting the slides.The formulations disclosed herein also can minimize quenching offluorescent dyes commonly used in cellular imaging applications.Formulations that include anti-fade reagents, for example, can resistquenching of dyes and particularly useful for imaging cells and tissuesstained with fluorescent probes. The unique combination of attributesdescribed above makes the described formulations particularly useful forlong term storage of stained, biological samples.

The refractive index matching (RIM) formulations provided herein caninclude one or more water-soluble components (e.g., a water-solublepolymer). The RIM formulations provided herein can include more than onewater-soluble component. In certain embodiments, the formulationincludes at least two water-soluble components. The water-solublecomponents can be contained in an aqueous medium, making theformulations suitable for use with biological specimens. The formulationcontaining the biological specimen can be imaged directly or dried priorto imaging to remove water from the formulation.

The RIM formulations provided herein exhibit a refractive index thatmatches the refractive index of substrates typically used in imagingapplications (e.g., microscope slide, such as those made from soda-limeglass or borosilicate glass) or immersion oil. The RIM properties of theformulations described herein can improve image quality, making thedescribed formulations ideal for use in imaging applications. RIMformulations provided herein exhibit an exceptionally high RI relativeto standard mounting formulations known to those skilled in the art.Typically, the refractive index of the disclosed formulations oncesolidified exceeds 1.45. Surprisingly, the disclosed formulations canprovide optically clear mounting formulations having a RI of 1.45 ofgreater. In certain embodiments, the RI of the mountant is 1.45 to 1.47.In other embodiments, mountants are provided with RI of 1.47 or greater(e.g., 1.47 to 1.50; or 1.50 to 1.52; or 1.52 to 1.54). In certainembodiments, the RI of the formulation is about 1.47 to about 1.53. Incertain embodiments, the RI of the mountant is about 1.50 to about 1.52.

For biological samples, such as tissue, that exhibit a RI range fromabout 1.50 to 1.52, it can be desirable to utilize a formulation thatcan provide a cured mountant having the same RI range. Thus, suitablewater-soluble components for use in the disclosed RIM compositions, oncedried, typically have a RI of 1.45 or greater, e.g., 1.45 to 1.60. Insome embodiments, the RI is 1.45 to 1.50. In other embodiments, the RIis 1.50 to 1.53. In other embodiments, the RI is 1.53 to 1.55. In yetother embodiments, the RI of the water-soluble components is 1.55 to1.60. Typically, the RI of the solution used to prepare the mountant isslightly lower than that of the cured mountant. Thus, also providedherein is a RIM solution, wherein the RIM solution has a refractiveindex of 1.33 or greater (e.g., 1.333 to 1.530; or 1.330 to 1.420).

The water-soluble component(s) can be a water-soluble polymer. Incertain embodiments, the formulation can further include a polyol. RIvalues for many types of polymers can be found in the literature. WhereRI data is unavailable, the RI for a particular polymer can be measuredor can be calculated based on Lorenz-Lorentz Theory using Equation 1,where n is the refractive index; and R_(LL) and V_(m) are the molarrefraction and molar volume of the polymer repeat unit, respectively.The RI calculated from Equation 1 is wavelength dependent and typicallyis reported at a wavelength of 589 nm (sodium d-line).

R _(LL) /V _(m)=(n ²−1)/(n ²+2)  (Equation 1)

R_(LL) and V_(m) values for numerous types of polymers can be found inthe literature or estimated from group contribution effects usingmethods known to those skilled in the art. The R_(LL)/V_(m) ratio for awater-soluble polymer suitable for use in a RIM formulation, asdescribed herein, typically ranges from about 0.27 to about 0.34. Incertain embodiments, a refractive index matching (RIM) formulation isprovided herein that includes a water-soluble polymer, wherein thepolymer has a molar refraction (R_(LL)) to molar volume (V_(m)) ratio of0.27 to 0.34, such that the RIM solution has a refractive index from1.45-1.60. In certain embodiments, formulations including awater-soluble polymer having a R_(LL)/V_(m) ratio in the range from0.284 to 0.314 provide RIM formulations having a refractive index fromabout 1.48 to about 1.54.

Water-soluble polymers and copolymers for use in the disclosed RIMformulations can be neutral or charged. For example, the water-solublepolymer can be a neutral (i.e., uncharged) polymer, such that it hasminimal interaction with dyes and/or cellular proteins present in thesample. Exemplary neutral, water-soluble polymers includepoly(acrylamide), poly(methacrylamide), poly(methyl vinyl ether),poly(vinyl pyrrolidone) (PVP), polyvinyl alcohol (PVA),poly(-ethyl-2-oxazoline), and poly(-methyl-2-oxazoline). Exemplaryacrylamide and methacrylamide-based polymers can include residues ofmonomer units, such as, e.g., N—R acrylamide or N—R methacrylamide,wherein R is methyl, ethyl, propyl, isopropyl or H;N,N-dimethylacrylamide, N,N-dimethylmethacrylamide,N,N-diethylacrylamide, N, N diethylmethacrylamide, N-2-hydroxypropylmethacrylamide), or a combination of these monomer units. In certainembodiments, the uncharged, water-soluble polymer is poly(N-methylmethacrylamide) (PMMAm). Formulations including neutral polymers, suchas, e.g., PMMAm, do not foam or form long-term air bubbles, as oftenoccur with existing commercial hard mount formulations, and, therefore,can be pipetted onto a sample with very little risk of bubble formation.Formulations containing neutral polymers can cure in less than 24 hours,making such polymers particularly useful for the preparation of RIMsolutions, as disclosed herein. For example, a formulation includingPMMAm can solidify in approximately 3 hours to provide a cured mountantwith an RI of 1.52.

Alternatively, the water-soluble polymer can be a charged polymer (e.g.,a polyelectrolyte). Charged polymers can be more soluble in water andmore viscous than neutral polymers of equivalent MW. Charged polymers,therefore, can have certain advantages when used in mountingformulations where such properties are desired. The charged polymer cancarry a positive or negative overall charge. Exemplary charged,water-soluble polymers include polyacrylic acid, polymethacrylic acid,poly(diallyldimethylammonium chloride), poly(sodium-4-styrenesulfonate),poly(ethyleneimine), poly(N,N-dimethylaminoethyl acrylate),poly(N,N-diethylethylamino acrylate), poly(allylamine),poly[bis(2-chloroethyl)ether-co-1,3-bis[3-(dimethylamino)propyl]urea],or poly(vinylsulfonic acid, sodium salt).

The molecular weight of the water-soluble polymer can be chosen based onthe desired properties of the mountant and its intended use. Forexample, the molecular weight of the polymer affects the viscosity ofthe formulation. Generally, the molecular weight is chosen to achieve aformulation that can be easily handled and yet sufficiently viscous suchthat it stays in place on the substrate. Polymers for use in thedisclosed formulations typically include a range of molecular weights.The lower end of the molecular weight (MW) range can be selected basedon the desired solubility of the polymer in the formulation, and theupper end of the MW range can be selected based on its ability to form ahardened surface upon drying. For example, if the MW is too low, thenthe formulations may not form a rigid film upon drying. In general, thewater-soluble polymer can have a weight average molecular weight ofabout 1 kDa to about 100 kDa, e.g., 1 kDa to 15 kDa; or 15 kDa to 20kDa; or 20 kDa to 40 kDa; or 40 kDa to 80 kDa; or 80 kDa to 100 kDa. Incertain embodiments, the weight average molecular weight of thewater-soluble polymer is about 48 kDa to about 80 kDa. Formulations thatinclude a higher molecular weight polymer (e.g., greater than about 15kDa can readily solidify under mild conditions that maintain theintegrity of the biological specimen. In some embodiments, the RIMformulation can implement a lower molecular weight, water-solublepolymer (e.g., less than 20 kDa) to provide a softer, more viscousmountant. A softer mountant can provide certain advantages depending onthe application and for mounting certain types of samples. For example,a softer mountant can be used with a specimen with thickness >200microns, when there is a need to minimize extended drying times, or whenrecovery of the sample from the formulation is desired.

The RIM formulations can further include one or more polyols. A “polyol”refers to a compound that includes two or more hydroxyl groups. Incertain embodiments, the polyol has 100 or more hydroxyl groups. Inother embodiments, the polyol has 1000 or more hydroxyl groups. Incertain embodiments, the polyol has 2500 or less hydroxyl groups. Apolyol can be included in the formulation to help plasticize the RIMpolymer, such that when the mounting film dries it does not become toobrittle or start cracking. A polyol that does not evaporate also canprovide some permanent volume to the film, thereby preventing the filmfrom becoming too thin. Ideally, the dried film maintains a volume thatis similar to the undried film. This can prevent deformation of aspecimen due to shrinkage of dried material Maintaining structure of thedried film is also important for sample archiving and storage at varioustemperatures (e.g. room temp, 4° C. and −20° C.). The polyol also shouldhave a RI close to that of the biological specimen and/or substrate orother components in the mounted system (e.g., 1.46 to 1.60). In someembodiments, the RI is 1.47 to 1.54. Exemplary polyols include glycerol,diglycerol, polyglycerol, polyvinyl alcohol, sugars such as mannitol,sorbitol, erythritol, thiodiethanol, thiodipropanol, and the like.Typically, the polyol is different from the water-soluble polymer.However, it is contemplated that in certain formulations the polymer andpolyol have the same composition. The molecular weights of the polymerand polyol can be the same or different. In a representative embodiment,e.g., PVA can be used as both the polyol and the water-soluble polymer,but the molecular weights of the two forms of PVA differ.

RIM formulations provided herein can include a combination of awater-soluble polymer and a polyol. The ratio of the water-solublepolymer to the polyol in the formulation can vary and can affect therefractive index of the dried hard mountant. Typically, formulationsintended for preparation of a hard mountant include a weight ratio ofwater-soluble polymer to polyol of 0.125 or greater. In certainembodiments, the weight ratio of water-soluble polymer to polyol rangesfrom 0.125:1 to 4:1. In certain embodiments, the weight ratio ofwater-soluble polymer to polyol is; or 0.125: 1 to 1:1; 1:1 to 2:1; 2:1to 3:1; or 3:1 to 4:1. In certain formulations, the weight ratio ofwater-soluble polymer to polyol is 1:1 to 3:1. In certain formulations,the weight ratio of water-soluble polymer to polyol is about 0.5:1.

The RIM formulation can further include an aqueous component (e.g.,water or a buffer), and the water-soluble polymer(s) and the polyol(s)are dissolved in the aqueous component. The aqueous component serves todissolve and/or hydrate the biological specimen and components of themounting formulation. Any biologically compatible buffer known to thoseskilled in the can be used in the formulations described herein, suchas, e.g., Tris, PBS, borate, and the like. Buffers that maintain the pHof the formulation above 7.4 are especially useful in certainformulations, such as, e.g., when fluorescent dyes are present.

The described formulations can improve sample transparency without theneed for an additional clearing agent. However, a clearing agentoptionally can be used for imaging tissue samples to further improveimage quality. Thus, in certain embodiments, the RIM formulationsdisclosed herein can be used in conjunction with a clearing agent. Forsamples including fluorescent materials, it can be preferable toimplement aqueous clearing agents given their compatibility with manyfluorophores and fluorescent proteins, as well as the instant RIMformulations. Exemplary clearing agents include, e.g., organicsolvent-based and aqueous detergent-based reagents, glycerol orthioglycerol solutions, mono- and polysaccharides (e.g., fructose andsucrose), urea solutions, and commercially available reagents.

RIM formulations provided herein resist formation of precipitates, incontrast to commonly used mountants that are known to form precipitateson samples over the course of as little as 2-3 days. RIM formulationsdescribed herein also prevent the sample and/or fluorescent labelingmaterials, if present, from photobleaching. Formulations suitable formounting and imaging biological specimens stained with a fluorescent dyeoptionally can further include an anti-fade reagent to minimizedegradation or photobleaching of the stained sample upon storage orinterrogation. Thus, in certain embodiments, the RIM formulationsprovided herein optionally include one or more antifade reagents and canbe included in the RIM formulation at about 1 wt.% or less. Antifadereagents are well known to those skilled in the art and includeanti-oxidants such as, e.g.,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 3-carboxyproxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate,ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine,1,4-diazabicyclo[2.2.2]octanel, other nitroxides, or a combinationthereof.

RIM formulations can further include additional components, such as,e.g., preservatives, to prevent degradation of the polymer and/or polyolduring prolonged storage and/or to prevent or minimize bacterial growth.Suitable preservatives include those that do not absorb a significantamount of visible light or lower the RI of the formulation.Representative preservatives include, e.g., sodium benzoate, benzoicacid or sodium azide, and are typically present in the formulation of aconcentration of less than 2% by weight. Additional components, such asdyes, also can be included in the RIM formulations provided herein. Incertain embodiments, the RIM formulation can include a fluorescent dyefor staining cells. In certain embodiments, the dye can stain thenucleus of a cell, such as, for example, Hoechst 33342 or DAPI.

Also provided herein are samples mounted on a substrate in a RIMmounting medium. For example, a biological specimen can be mounted on asubstrate and embedded in a RIM formulation, as described herein. Asubstrate can be any material having a surface suitable for supportingor containing the RIM solution. For example, the substrate can have asurface that is smooth or rough and can be flat or contain a cavity ordepression (e.g., a well for containing the formulation). In certainembodiments, the substrate has a relatively smooth and non-poroussurface, such that RIM solution does not become absorbed into thesubstrate. Substrates can be optically transparent or non-tranparent,and can be made from a variety of materials, depending on the ultimateend use. For microscopy applications, e.g., the substrate can beoptically transparent. Substrates can be made from any appropriatematerial, including, e.g., glass or a polymer. Representative substratesinclude but are not limited to those commonly used in optical andimaging applications such as, e.g., a microscope slide, cuvette, well,coverslip or dish. In certain embodiments, the mounted sample canincludes a transparent substrate (e.g., a microscope slide), abiological specimen (e.g., tissue), and a solidified RIM formulationsurrounding the sample and adhering it to the substrate. Optionally, acoverslip can be disposed over the solidified, mounted sample. Theamount of solidified formulation on the mounted slide is sufficient tosurround the biological specimen and adhere it to the substrate and/orcoverslip.

Mounted biological samples, as disclosed herein, can be stable for manymonths (e.g., 5 months or more) when stored at room temperature or lesswithout degrading or fading the sample or fluorescent stains, ifpresent.

The sample can be any biological material including, but not limited to,tissues, cells, blood, and the like. The biological sample can bestained with a fluorescent dye(s) or fluorescent protein prior tomounting on the substrate. Fluorescent proteins suitable for stainingbiological samples are well known in the art and include, e.g., withoutlimitation, GFP, RFP, mCherry, and the like. Commonly used fluorescentdyes for biological specimens include, e.g., boron dipyrromethenes(4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes), cyanines, xanthenes,sulfonated pyrenes, rhodamines, coumarins, and derivatives thereof.Exemplary organic dyes include BODIPY dyes, coumarins (e.g., PACIFICBLUE, PACIFIC GREEN and PACIFIC ORANGE (available from Thermo FisherScientific; Waltham, Mass.)), rhodamines, rhodol, fluorescein,thiofluorescein, aminofluorescein, carboxyfluorescein,chlorofluorescein, methylfluorescein, sulfofluorescein, aminorhodol,carboxyrhodol, chlororhodol, methylrhodol, sulforhodol; aminorhodamine,carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine,silicon rhodamine, and thiorhodamine, cyanine, indocarbocyanine,oxacarbocyanine, thiacarbocyanine, merocyanine, cyanines (e.g., cyanine2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7),oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole,benzoxadiazole, pyrene derivatives, cascade blue, oxazine derivatives,Nile red, Nile blue, cresyl violet, oxazine 170, acridine derivatives,proflavin, acridine orange, acridine yellow, arylmethine derivatives,xanthene dyes, sulfonated xanthenes dyes, sulfonated pyrenes, auramine,crystal violet, malachite green, tetrapyrrole derivatives, porphyrin,phtalocyanine, bilirubin and bis-benzimides (Hoechst stains). In certainembodiments, the organic dye is a near-infrared dye, such as, e.g.,CY5.5 (GE Healthcare Life Sciences; Pittsburgh, Pa.), IRDYE 800 (Li-Cor;Lincoln, Nebr.), DYLIGHT 750 (Thermo Fisher Scientific) or indocyaninegreen (ICG), or a cyanine dye, such as, e.g., cyanine 2, cyanine 3,cyanine 3.5, cyanine 5, cyanine 5.5, cyanine 7. In certain embodiments,the organic dye is a xanthene or sulfonated xanthenes dyes, such asthose commercially available under the tradenames ALEXA FLUOR 594, ALEXAFLUOR 633, ALEXA FLUOR 647 and ALEXA FLUOR 700 (Thermo FisherScientific). Additional examples of suitable commercially available dyesinclude ALEXA FLUOR 405, ALEXA FLUOR 488 and ALEXA FLUOR PLUS secondaryantibodies (Thermo Fisher Scientific).

The mounted sample can be interrogated directly or can be cured usingmethods described herein to provide a biological specimen that isembedded in a solidified mountant. Advantageously, the refractive indexof the mounted and cured sample matches the RI of the substrate (e.g.,microscope slide) and coverslip, and usually exceeds 1.47 and typicallyranges from 1.48 to 1.54. For example, the solidified mountantcontaining the specimen can have a refractive index can range from 1.48to 1.54 (e.g., 1.48 to 1.50; or 1.50 to 1.52; or 1.52 to 1.54).

Further provided herein are methods of mounting a biological specimen ona substrate. A representative method includes depositing the biologicalspecimen on the substrate and then contacting the biological specimenwith the RIM solution to provide a mounted sample. Once mounted, thesample can be interrogated directly (e.g., imaged using a microscope).However, the sample is typically covered (e.g., with a coverslip) forprotection during use and subsequent storage. The formulation containingthe biological specimen then can be imaged or subsequently dried. Sampledrying (also referred to herein as “curing”) removes water from the RIMformulation, thereby resulting in hardening of the mountant. Forformulations that include a water-soluble polymer, removal of waterprovides a solidified formulation that is referred to herein as a “hardmountant” or “hard-mount” formulation. It should be appreciated that ahardened mountant still can maintain a certain level of viscosity and/orelasticity, such that it does not become too brittle or cracked.

Depending on the type of formulation and the desired level of curing,the mounted sample can be subjected to a range of different curingregimens. The temperature and time required to cure a particularformulation depends on various factors, including, e.g., the type,molecular weight and concentration of the water-soluble component in theformulation, as well as the desired extent of curing. In addition, theconditions for curing depend on whether the sample is covered with acoverslip or open to the air.

Drying can be conducted at ambient (e.g., room) temperature or at anelevated temperature. In certain embodiments, curing occurs at atemperature below about 40° C. to prevent damage to the biologicalspecimen and/or degradation to the water-soluble components in theformulation. In formulations containing a water-soluble polymer, thepolymer can solidify in 24 hours or less; or in some cases 10 hours orless; or even 4 hours or less. In certain formulations including awater-soluble polymer with a molecular weight of 20 kDa or greater, asdisclosed herein, drying can be achieved at a temperature below about40° C. in 4 hours or less in the absence of a coverslip.

The mounting formulations provided herein can be used in varioushistochemistry, immunochemistry and cytochemistry applications,including, but not limited to mounting of hematological, histological,and cytological samples on microscope slides, including tissue andblood. The described RIM formulations can be used to provide specimensmounted on substrates such as samples of blood, cells, tissue or otherbiological fluids or materials, including but not limited to, materialsthat have been stained to facilitate microscopic examination forresearch and/or diagnostic purposes. A mounted biological specimenprepared according to the methods disclosed herein can be visualizedusing imaging techniques that are well-known in the art. Imaging can beperformed, e.g., using an optical microscope. The high RI and opticalclarity of the mounting formulations described herein allow biologicalsamples to be imaged to greater depths (e.g., 100 μm or less; e.g. 1-100μm) and to higher resolution than when using standard mountants. Forexample, fluorescently labeled targets are detectable down to 100 μm,and resolution is maintained to a similar depth.

Samples can be prepared using standard ICC/IHC protocols. Additionaldehydration in alcohols (e.g., methanol and ethanol) prior to mountingcan reduce drying time. Drying in the presence of a desiccant aftersample mounting also can speed drying time. The sample can be coatedwith mountant to cover it and dried without a coverslip. The mountedsample then can be imaged directly without a coverslip or covered with asmall volume of glycerol and covered prior to imaging. Any suitablebiological sample can be evaluated using the methods disclosed herein,including, but not limited to solution- or suspension-based samples andtissue samples. In certain embodiments, the sample includes cells or adigested cells and fragments thereof. The cells can be dead (e.g.,fixed) or live. Additional examples of biological materials that can betreated with the RIM formulations disclosed herein include, a 3D cellculture (spheroid/organoid) or a whole organism (e.g. fruit fly, worm,zebra fish).

The RIM formulations described herein can be used to identify featuresof different materials, including, without limitation, plant, microbial,animal, earth, blood and plasma samples, and other types of non-livingorganic and inorganic materials, including but not limited to soilparticles and geological samples, without losing clarity, definition orresolution of the objective structures.

In order to identify different components in a cell, stains can be usedto provide contrast between particular structures based on theirchemical composition. The RIM solutions disclosed herein do notinterfere with the fluorescent dyes typically used for staining cells.Thus, in certain embodiments, the biological specimen can be labeledwith a fluorescent dye including, but not limited to, those disclosedherein. For example, the sample can include cells that express surfaceantigens (e.g., cell surface receptors) that can be recognized by andbind to specific affinity molecules (e.g., antibodies). Cells can betreated with a conjugate that includes a fluorophore attached to anaffinity molecule (e.g., an antigen) under conditions for binding theantigens on the surface of the cells to the affinity molecule to form acell labeled with the conjugate. Alternatively, the fluorophore can becontained within the cells, e.g, in the cytoplasm or within an organelleor cellular membrane. Further, RIM formulations provided herein can beused in imaging of stained cells and tissues, and are particularlyuseful in fluorescent immunohistochemistry (IHC) applications.

A general method of preparing a specimen involves soaking the specimen,which can be optionally stained, in a sufficient quantity of themounting solution to fully immerse the specimen within the solution. Thespecimen then is applied to the substrate (e.g., a microscope slide,cuvette, or well). Optionally, the mounted specimen can be dried (e.g.at room temperature up to about 40° C.) until hardened. A cover slip canbe applied over the specimen prior to or after hardening of the sample.Cured, mounted samples can be stored indefinitely. Alternatively, themounted sample can be interrogated, e.g., visualized under a microscope.The mounting formulations are compatible with fluorescent microscopesand objectives, such as epi-fluorescent, wide-field, confocal,stimulated emission depletion (STED) and structured illuminationmicroscopes (SIM). Due to the superior optical properties of the RIMformulation, the specimen appears transparent, allowing visualization ofcells and deeper layers of tissues without losing clarity.

The described RIM formulations also are ideally suited for mountingtissue samples on a substrate. Whole tissue mounting and imaging hasbeen challenging to date, because sample thickness, presence ofextracellular materials, and reagent penetration can result inbackground fluorescent and poor image resolution, and decreased imagingdepth. The described formulation can decrease the refraction of light asit passes through a coverslip and tissue sample allowing higherresolution and the ability to image to a greater depth. Because thedisclosed formulations improve sample transparency, they areparticularly advantageous for use with tissue samples.

A representative method is provided for mounting a tissue sample on amicroscope slide using a RIM formulation, as described herein. Thetissue can be a sectioned or whole sample that can be fixed or unfixed.In general, the tissue sample is deposited onto the surface of amicroscope slide and a RIM formulation, as disclosed herein, is appliedto the sample. The mounted sample then is dried to remove excess water,such that the formulation solidifies. The amount of the mounting mediumused in the method can vary but is generally chosen to wet the surfaceof the tissue. Excess mounting medium can be removed using knownmethods, if desired. Once mounted onto the slide surface, the mountedsample can be dried, with or without the application of heat (e.g., atroom temperature or at an elevated temperature). Drying can beaccelerated by using a vacuum pump and/or desiccant. Once dried, thetissue firmly adheres to the slide and the sample will be embeddedwithin the mountant. If needed, an additional amount of the formulationcan be added, and the drying process can be repeated.

Further provided herein are kits for mounting a biological sample to asubstrate for subsequent imaging and/or storage of the sample. Thus, inyet another aspect, a kit for mounting a biological specimen on asubstrate is provided that includes a refractive index matching (RIM)solution, as disclosed herein; and instructions for mounting abiological specimen on the substrate. Additional components optionallycan be included in the kit, including, e.g., a co-mountant, such asglycerol.

The following examples are included to demonstrate certain embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor(s) to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the scope of theinvention.

EXAMPLES

The examples provided herein utilize the following general methodsunless indicated otherwise. Refractive indices of all mountantformulations before and after drying were measured with an ABBE-3LRefractometer (Thermo Fisher Scientific) equipped with a lamp operatingwavelength sodium D line (589.3 nm). Molecular weights of poly(N-methylmethacrylamide) (PMMAm) are reported as weight average molecular weight(M_(w)), as measured by standard gel permeation chromatography (GPC)methods using 5 mM LiBr methanol/dimethylformamide (50/50 v/v %) as aneluent, TSkgel® HH, organic size exclusion columns (TOSOH BioscienceLLC, King of Prussia, Pa.) with Wyatt Technology Corporation's miniDAWN®TREOS® multi-angle light scattering (MALS) and Optilab® T-rEXdifferential refractometer detectors (Santa Barbara, Calif.).

Example 1 Measuring Refractive Indices of Mountant Samples

Refractive index measurements on dried samples were measured bycarefully spreading 300 μL of sample onto the refractometer prism andthen allowing the mountant to dry up to 24 hours. Refractive index wasmeasured once the mountant solution was dried into a transparent film.

Example 2 Mounting Procedure

Biological specimens (e.g., cultured monolayer cells and tissue sectionswith thickness of less than 30 μm) are mounted onto a substrate byapplying a sufficient amount of mountant, typically 1 to 3 drops (30 to100 μL), to a specimen disposed on a substrate until fully covered orimmersed. The biological specimen can be unstained or can be stainedprior to mounting using procedures well known to those in the art. Thespecimen then is covered with an 18 mm×18 mm coverslip and any excessmountant is wiped or pipetted away from the edges of the coverslip. Themounted specimen is allowed to dry for 24-96 hours at room temperature,although temperatures as high as 40° C. are acceptable. After themounted specimen is completely dried, the specimen can be imaged usingfluorescent microscopy techniques that are well known to the personskilled in the art. Alternatively, the mounted specimen can be driedprior to applying a coverslip. Drying without a coverslip can acceleratethe drying of the hard set mountant. Without a coverslip, drying cantake about 30 minutes at 40° C. to about 4 hours at room temperature.After the mountant is dry, a coverslip can be applied over the mountedspecimen by placing a contact liquid such as water, more mountant,ethanol or glycerol on the dried mountant and then placing a coverslipover the specimen.

The following protocol can be used for mounting biological specimensthat are 30-100 μm and thicker. Allow the mountant to warm to roomtemperature for 2 hours before mounting coverslips. Remove excess liquidfrom the sample by gently tapping the edge of the coverslip or slide ona laboratory wipe. For slide-mounted specimens, apply 2-3 drops or60-100 μL of the mountant directly to the specimen, then carefully lowera coverslip onto the mountant to avoid trapping any air bubbles. Forspecimens stained in well plates or culture dishes, carefully movesample to a microscope slide. The addition of 10 μL of mountant to theslide can assist with manipulating the sample into place. For 3-Dcultured cells or spheroids, move 3-D cultures or spheroids to amicroscope slide using a lmL pipette with the end of the tip removed.Place the 3-D culture with buffer on a microscope slide prepared with anappropriate spacer to ensure integrity of the sample. The spacer shouldallow sufficient room for the sample while minimizing the volume ofmountant required. Spacers allowing open edges decrease the curing timeof the sample. If needed, gently tap the coverslip to remove airbubbles. If the mountant does not fill out to the edges of thecoverslip, apply additional mountant under the coverslip using apipette. Gently tap to remove air bubbles from around the sample.Failure to sufficiently cover the sample can lead to mountantcontraction and reduce the imaging area. Place the mounted sample on aflat, dry surface, and allow it to cure at room temperature in the dark.For optimal results, allow sample to cure for at least 48 hours.

Specimens 30-100 μm and thicker can be cured more quickly using thefollowing protocol. Allow the mountant to warm to room temperature for 2hours before mounting coverslips. Remove excess liquid from the sampleby gently tapping the edge of the coverslip or slide on a laboratorywipe. For slide-mounted specimens, apply 2-3 drops or 60-100 μL of themountant directly to the specimen. Carefully tilt the slideback-and-forth to distribute the mountant evenly over the specimen. Aimto spread 2-3 drops or 60-100 μL of mountant over an area of 18 mm×18mm. The edge of a pipette tip can be used to gently assist in removingany bubbles and spreading the mountant. For specimens stained in wellplates or culture dishes, carefully move the sample to a microscopeslide. The addition of 10 μL of mountant to the slide can assist withmanipulating the sample into place. For 3-D cultured cells or spheroids,move 3-D cultures or spheroids to a microscope slide using a 1-mLpipette with the end of the tip removed. Place the 3-D culture withbuffer on a microscope slide prepared with an appropriate spacer toensure integrity of the sample. The spacer should allow sufficient roomfor the sample while minimizing the volume of mountant required. Removeexcess buffer from the culture, then apply 2-3 drops or 60-100 μL of themountant directly to the specimen depending on the spacer height andsurface area. Do not apply a coverslip at this point. Allow the sampleto cure without a coverslip for 16-24 hours at room temperature,protected from light. After 16-24 hours, apply 10 μL of glycerol acrossthe top of the cured mountant and specimen. Apply a coverslip and pressinto place, tapping to remove bubbles, if present. Allow the coverslipto cure into place for 1-3 hours or until the coverslip no longer moves.

Example 3 Formulation of Mounting Media with Polyvinyl Pyrrolidone (PVP)

Mounting media was formulated by adding 0.50 g of glycerol (FisherScientific, Fair Lawn, N.J.), 1.00 g of PVP (M_(w)=55,000 g/mol; TCIAmerica Portland, Oreg.), 61 mg of2-amino-2-(hydroxymethyl)-1,3,propanediol (TRIS base; Sigma-Aldrich, St.Louis, Mo.) and 3.8 mL of deionized water to a 20 mL scintillation vialequipped with a magnetic stir bar. The solution was stirred at 500 rpmin a water bath at 60° C. and heated until complete dissolution of PVPwas observed. After dissolution of the polymer, the vial was allowed tocool to room temperature prior to use. The RI was measured as describedin Example 1 (see, Table 1, Sample 2).

Example 4 Formulation of Mounting Media with Polyvinyl Alcohol (PVA)

Mounting media was formulated by adding 0.50 g of glycerol, 61 mg ofTRIS base, 1.00 g of PVA (M_(w)˜23,000 g/mol; Sekisui AmericaCorporation, Secaucus, N.J., USA) and 3.8 mL of deionized water to a 20mL scintillation vial equipped with a magnetic stir bar. The solutionwas stirred and heated as described in Example 3 until completedissolution of PVA was observed. After dissolution of the polymer thevial was allowed to cool to room temperature prior to use. The RI wasmeasured as described in Example 1 (see, Table 1, Sample 8).

Example 5 Formulation of Mounting Media with Poly(N-methylmethacrylamide) (PMMAm)

Mounting media was formulated by adding 0.50 g of glycerol, 61 mg ofTRIS base, 1.00 g of PMMAm (M_(w)=100,000 g/mol) and 3.8 mL of deionizedwater to a 20 mL scintillation vial equipped with a magnetic stir bar.The solution was stirred and heated as described in Example 3 untilcomplete dissolution of PMMAm was observed. After dissolution of thepolymer the vial was allowed to cool to room temperature prior to use.The RI was measured as described in Example 1 (see, Table 1, Sample 14).

Example 6 Formulation of Mounting Media with Varying Polymer/GlycerolWeight Ratios

Mounting media were formulated using PVP, PVA and PMMAm as thewater-soluble polymer, as described in Examples 3-5. In addition, theweight ratio of polymer to glycerol was varied from 0.5 to 2.0. Thefinal volume of the mountant was diluted with DI water to a constantvolume of 5 mL. The solution was stirred and heated as described inExample 3 until complete dissolution of the polymer was observed. Afterdissolution of the polymer, the vial was allowed to cool to roomtemperature prior to use. The RI's were measured as described in Example1 (see, Table 1, Samples 1, 2, 3 and 4 for PVP; Samples 7, 8 and 10 forPVA; and Samples 14, 15, 17, 18 and 20 for PMMAm). Referring to the datafor Samples 1, 2, 3, 4, 7, 8, 10, 14, 15, 17, 18 and 20 in Table 1, theRI of the dried mounting media increased significantly as the ratio ofpolymer to glycerol increased, which was expected because the RIs of thetested polymers reported in the literature (i.e., 1.530 PVP; 1.50 PVA;and 1.540 PMMAm) exceed that of pure glycerol (1.4722 @ 25° C.).However, it was found that once the polymer concentration exceeds acertain threshold value, the physical and optical properties of thedried mountant degrade considerably and, depending on the polymer, caneven result in a film that becomes unusable for microscopy applications(see, e.g., Sample 23). However, too much polyol in a formulation canresult in a dried material that is greasy or oily and the coverslip willnot adhere well to the sample. For certain polymers this threshold valueis about 0.125:1 or greater (e.g., PVP and PVA), and only minutequantities of polymer are required to produce a useable, high RIM film.For other polymers, however, too much polymer in the formulation canlead to poor film quality and have other disadvantages. For formulationsimplementing PMMAm, for example, the presence of some polyol in theformulation helps to plasticize the polymer, so that it does not becometoo brittle or crack, especially when stored at lower temperatures.Thus, for PMMAm-based formulations, the weight ratio of thewater-soluble polymer to the polyol is about 4:1 or less (e.g., 0.125:1to 4:1). Regardless of polymer type, polyol also can impart some levelof permanent volume to the dried mountant and can prevent the biologicalsample (e.g, cells) from compressing too much. For example, even thoughPVA films without polyol do not typically crack upon drying, the filmshrinks so much that the cells become compressed.

Example 7 Formulation of Mounting Media with Varying Polymer/DiglycerolWeight Ratios

Mounting media was formulated as described in Example 6 with theexception that diglycerol (Alfa Aesar, Tewksbury, Mass.) was used inplace of glycerol as the polyol. The weight ratios of polymer todiglycerol were varied from 0.5 to 2.0. The final volume of the mountantwas diluted with DI water to a constant volume of 5 mL. The solution wasstirred and heated as described in Example 3 until complete dissolutionof polymer was observed. After dissolution of the polymer the vial wasallowed to cool to room temperature prior to use. The RIs were measuredaccording to the procedure described in Example 1 (see, Table 1, Samples5 and 6 for PVP; Samples 9, 11 and 12 for PVA; and Samples 13, 16, and19 for PMMMAm). Referring to Table 1, the RI of the dried mounting mediaincreased as the ratio of polymer to diglycerol increased, because theRIs of the tested polymers exceed that for pure diglycerol (1.4850 @ 20°C.). In addition, mounting media prepared with diglycerol had slightlyhigher RIs than when prepared using glycerol.

Example 8 Formulation of Mounting Media with Varying Polymer MolecularWeight

Mounting media were formulated as described in Examples 3-5 with theexception that the weight of the polymer was varied as follows: PVP(10,000 g/mol to 55,000 g/mol); PVA (13,000 g/mol to 31 000 g/mol); andPMMAm (33,000 g/mol to 100,000 g/mol). The weight ratios of polymer topolyol were varied from 0.5 to 2.0. The final volume of the mountant wasdiluted with DI water to a constant volume of 5 mL. The solution wasstirred and heated as described in Example 3 until complete dissolutionof polymer was observed. After dissolution of the polymer, the vial wasallowed to cool to room temperature prior to use. The RIs were measuredaccording to the procedure described in Example 1 (see, Table 1, Samples1-6 for PVP; Samples 7-12 for PVA; and Samples 13-20 for PMMMAm).Interestingly, the molecular weight of the polymer had no statisticallysignificant effect on the RI when the polyol type and polymer to polyolratio were held constant.

Example 9 Formulation of Mounting Media with Polymer Only

Mounting media was formulated as described in Examples 3-5 with theexception that glycerol or diglycerol additive were omitted from theformulations. The final volume of the mountant was diluted with DI waterto a volume of 5 mL. The solution was stirred and heated as described inExample 3 until complete dissolution of polymer was observed. Afterdissolution of the polymer the vial was allowed to cool to roomtemperature prior to use. The RI's were measured according to theprocedure described in Example 1 (see, Table 1, Samples 21-23). The RIof Sample 23 was not measurable (NM) due to the cracking of the driedmountant. The data for samples absent glycerol or diglycerol indicatethat the presence of a polyol in the formulation can prevent the filmfrom becoming too brittle or shrinking too much, likely because that thepolyol plasticizes or softens the dried polymer film.

TABLE 1 Refractive indices of various mountant formulations upon dryingfor 24 h at room temperature Polymer Weight Polymer/Polyol AverageMolecular Refractive Weight Ratio Weight (M_(w)) Index Sample PolymerPolyol (g/g) (g/mol) (×1000) @ 21° C. 1 PVP Glycerol 2 10 1.5037 2 PVPGlycerol 2 55 1.5000 3 PVP Glycerol 0.5 40 1.4757 4 PVP Glycerol 1 101.4830 5 PVP Diglycerol 0.5 55 1.4797 6 PVP Diglycerol 2 40 1.5090 7 PVAGlycerol 2 13 1.4937 8 PVA Glycerol 2 23 1.4927 9 PVA Diglycerol 2 311.4992 10 PVA Glycerol 0.5 31 1.4687 11 PVA Diglycerol 0.5 13 1.4712 12PVA Diglycerol 2 23 1.4988 13 PMMAm Diglycerol 2 33 1.5172 14 PMMAmGlycerol 2 100 1.5154 15 PMMAm Glycerol 1 33 1.4972 16 PMMAm Diglycerol2 100 1.5192 17 PMMAm Glycerol 0.5 33 1.4813 18 PMMAm Glycerol 2 661.5132 19 PMMAm Diglycerol 0.5 66 1.4823 20 PMMAm Glycerol 2 33 1.513421 PVP None N/A 40 1.5158 22 PVA None N/A 23 1.4986 23 PMMAm None N/A 66NM

Example 10 Formulation of Mounting Media with Polymer and AntifadeReagent

Mounting media was formulated by adding 0.55 g of diglycerol, 1.10 g ofPMMAm (M_(w)=60,000 g/mol), 122 mg of2-amino-2-(hydroxymethyl)-1,3,propanediol (TRIS base) and 5.0 mL ofdeionized water to a 20 mL scintillation vial equipped with a magneticstir bar. The solution was stirred and heated as described in Example 3until complete dissolution of PMMAm was observed. After completedissolution of the polymer, 9.7 mg of benzoic acid (7.94 mmol) and 19 mgof sodium sulfite (15 mmol) were added as a preservative and antifadereagent, respectively. The solution was stirred for an additional 30minutes at 60° C. until all the benzoic acid was dissolved. Oncedissolved, the vial was cooled to room temperature and the volume wasdiluted up to 10 mL with DI water. The RI was measured according to theprocedure described in Example 1 (RI=1.5146 @ 21° C. after 24 h). The RIvalue for this formulation is most similar to Sample 18 or Sample 13presented in Table 1. Example 8 indicates that MW does not statisticallyinfluence RI values and that the use of diglycerol versus glycerolslightly increases RI values. In comparison to Sample 13 and Sample 18,an RI value of 1.5146 for the current sample suggests that the additionof antifade and preservative reagents have a negligible effect, if any,on the final RI value.

Example 11 Refractive Index Measurements over Time

Refractive indices were monitored over a 5 day period to track thechange in RI as a function of time. Various mounting formulations wereevaluated as described in Example 1, and measurements were repeated atvarious time points over a 5 day period. The refractive index increaseddramatically over the first 5 h and then plateaued and remained similarafter 24 h (see, FIG. 1 for Sample 8 and 13).

Example 12 Mounting Sub-Micron Fluorescent Microspheres for AxialResolution Measurements Using Confocal Microscopy

The axial resolution at various focal depths for mounting formulationsdisclosed herein is detected with a confocal microscope using the pointspread function of sub-resolution fluorescent microspheres. 1.7 μL of175 nm green fluorescent microspheres (505/515 excitation/emission) froma PS-Speck™ Microscope Point Source Kit (Thermo Fisher Scientific,Waltham, Mass.) are dispersed in 200 μL of mountant sample and thensonicated for 20 minutes. After sonication, 100 μL of each sample ispipetted onto ethanol cleaned 18 mm by 18 mm coverslips and spread overthe entire area by tilting the coverslips back and forth with forceps.The mounting media samples were dried on the coverslips for 1 h at 40°C. Once dried, coverslips were immobilized onto standard microscopeslides by placing 10 μL of glycerol onto the slides and then gentlyplacing each coverslip onto the glycerol droplet. Coverslips wereallowed to immobilize on the slide for ˜20 minutes at room temperature.

Axial and lateral resolutions for each sample were determined byconfocal microscopy. Z-stacks of individual microspheres were collectedon a Zeiss™ LSM 710 confocal microscope using a Plan-Apochromat 63x/1.4NA Oil objective, sampling at a rate of 42 nm in x, y and 100 nm in thez dimensions. Prior to imaging each sample, the objective was firstcovered with Carl Zeiss Immersol™ 518F immersion oil (Carl Zeiss, Inc.,Thornwood, N.Y.) having a RI of 1.518 (measured with an e-line (546 nm)at 23° C.). For each sample, embedded microspheres were imaged at focaldepths ranging from the coverslip down to 100 μm. Three microsphereswere imaged at every focal depth for each sample at different locationsacross the entire area of the mounted sample (i.e. left, center andright). Axial (z) and lateral (x, y) resolutions for each sample werecalculated using the ImageJ MetroloJ plugin. Plotted data (FIG. 2 andFIG. 3) shows axial and lateral resolutions as a function of focal depthfor Sample 8, 10 and 13. As seen in FIG. 2, axial resolution is improved(i.e lower values) the closer the RI is to the RI of immersion oil.Improved axial resolution follows Sample 13 (1.5172)>Sample 8(1.4927)>Sample 10 (1.4687), where Sample 13 has 1.54 times improvedaxial resolution in contrast to Sample 10 at a 100 μm focal depth. Asexpected, there is little to no difference in lateral resolution betweeneach sample at each focal depth (FIG. 3) Tuning of the RI to achievemaximal axial resolution, while maintaining integrity of the driedmounting media, through the manipulation of the polymer type andpolymer/polyol ratio to match that of the RI of immersion oil isdemonstrated in this Example.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following clauses andclaims, the terms used should not be construed to limit the clauses andclaims to the specific embodiments disclosed in the specification andthe claims, but should be construed to include all possible embodimentsalong with the full scope of equivalents to which such clauses andclaims are entitled. Accordingly, the clauses and claims are not limitedby the disclosure. Embodiments may be in accordance with followingnumbered clauses:

-   -   1. A refractive index matching (RIM) solution, comprising:    -   a) a water-soluble polymer, wherein the polymer has a molar        refraction (R_(LL)) to molar volume (V_(m)) ratio of 0.27 to        0.34;    -   b) a polyol, wherein the weight ratio of the water-soluble        polymer to the polyol is 0.318:1 to 4:1; and    -   c) water or a buffer, wherein the RIM solution has a refractive        index of 1.36 or greater.    -   2. The RIM solution of clause 1, wherein the refractive index is        1.60 or less.    -   3. A method of mounting a biological specimen on a substrate,        comprising:    -   depositing the biological specimen on the substrate; and    -   contacting the biological specimen with the RIM solution of        clause 1 or clause 2 to provide a mounted sample.    -   4. The method of clause 3, further comprising drying the mounted        sample, whereby the water-soluble polymer solidifies to provide        a solidified polymer.    -   5. The method of clause 4, wherein the water-soluble polymer        solidifies in about 48 hours or less at room temperature; or 24        hours or less at room temperature; or in about 4 hours or less        at a temperature of about 40° C.    -   6. The method of clause 4, wherein the refractive index of the        solidified polymer exceeds 1.47 (e.g., 1.48 to 1.54; or 1.50 to        1.525).    -   7. The method of any one of the preceding clauses, further        comprising visualizing the biological specimen on the substrate        with a microscope.    -   8. A solidified mountant, comprising a water-soluble polymer,        wherein the polymer has a molar refraction (R_(LL)) to molar        volume (V_(m)) ratio of 0.27 to 0.34, and a polyol, wherein the        weight ratio of the water-soluble polymer to the polyol is        0.318:1 to 4:1, and wherein the solidified mountant has a        refractive index of 1.48 or greater.    -   9. The solidified mountant of clause 8, wherein the weight ratio        of the water-soluble polymer to the polyol is 0.5:1 or greater.    -   10. The solidified mountant of clause 8 or 9, wherein the        refractive index is greater than 1.47.    -   11. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer is        uncharged.    -   12. The RIM solution of clause 11, wherein the uncharged,        water-soluble polymer is a poly(acrylamide),        poly(methacrylamide), poly(methyl vinyl ether), poly(vinyl        pyrrolidone) (PVP), polyvinyl alcohol (PVA),        poly(2-ethyl-2-oxazoline), or poly(2-methyl-2-oxazoline).    -   13. The RIM solution of clause 11, wherein the uncharged,        water-soluble polymer comprises a monomer residue of N-R        acrylamide or N-R methacrylamide, wherein R is methyl, ethyl,        propyl, isopropyl or H; N,N-dimethylacrylamide,        N,N-dimethylmethacrylamide, N,N-diethylacrylamide, N, N        diethylmethacrylamide; N-2-hydroxypropyl methacrylamide); or a        combination thereof.    -   14. The RIM solution or solidified mountant of clause 11,        wherein the uncharged, water-soluble polymer is poly(N-methyl        methacrylamide) (PMMAm).    -   15. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer is charged.    -   16. The RIM solution or solidified mountant of clause 15,        wherein the charged, water-soluble polymer is polyacrylic acid,        polymethacrylic acid, poly(diallyldimethylammonium chloride),        poly(sodium-4-styrenesulfonate), poly(ethyleneimine),        poly(N,N-dimethylaminoethyl acrylate),        poly(N,N-diethylethylamino acrylate), poly(allylamine),        poly[bis(2-chloroethyl)ether-co-1,3-bis[3-(dimethylamino)propyl]urea],        or poly(vinylsulfonic acid, sodium salt).    -   17. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer has a        weight average molecular weight of about 1 kDa to about 100 kDa.    -   18. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer has a        weight average molecular weight of about 1 kDa to about 20 kDa.    -   19. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer has a        weight average molecular weight of about 20 kDa to about 100        kDa.    -   20. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer has a        weight average molecular weight of about 48 kDa to about 80 kDa.    -   21. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the refractive index is 1.48-1.50; or        1.50 to 1.52; or 1.52 to 1.54.    -   22. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the refractive index of the RIM        solution or solidified mountant matches the refractive index of        soda-lime glass, borosilicate glass, or immersion oil.    -   23. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the water-soluble polymer is not a        polyol.    -   24. The RIM solution or solidified mountant of any one of the        preceding clauses, wherein the polyol is polyvinyl alcohol,        glycerol, diglycerol, polyglycerol, mannitol, sorbitol,        thiodiethanol, thiodipropanol, or a combination thereof.    -   25. The RIM solution or solidified mountant of any one of the        preceding clauses, further comprising an anti-oxidant.    -   26. The RIM solution or solidified mountant of clause 25,        comprising 1% by weight or less of the anti-oxidant.    -   27. The RIM solution of clause 26, wherein the anti-oxidant is        6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid,        3-carboxy proxyl, hydroquinone, mequinol, sodium sulfite, sodium        erythorbate, ascorbic acid, propyl gallate, caffeic acid,        paraphenylenediamine, 1,4-diazabicyclo[2.2.2]octane, or a        combination thereof.    -   28. A mounted biological specimen, comprising a biological        specimen disposed on a substrate, wherein the biological        specimen is embedded in the solidified mountant of any one of        the preceding clauses.    -   29. A kit for mounting a biological specimen on a substrate,        comprising:    -   a) the refractive index matching (RIM) solution of any one of        the preceding clauses; and    -   b) instructions for mounting the biological specimen on the        substrate.    -   30. The method, mounted biological specimen or kit of any one of        the preceding clauses, wherein the substrate is a microscope        slide, cuvette, well or dish.    -   31. The method, mounted biological specimen or kit of any one of        the preceding clauses, wherein the biological specimen is a        cell, tissue, 3D cell culture (e.g., spheroid/organoid), or a        whole organism (e.g. fruit fly, worm, zebra fish).    -   32. The method, mounted biological specimen or kit of any one of        the preceding clauses, wherein the biological specimen is        labeled with a fluorescent dye or fluorescent protein.

1. A refractive index matching (RIM) solution, comprising: a) a water-soluble polymer, wherein the polymer has a molar refraction (RLL) to molar volume (Vm) ratio of 0.27 to 0.34; b) a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.318:1 to 4:1; and c) water or a buffer, wherein the RIM solution has a refractive index of 1.36 or greater.
 2. The RIM solution of claim 1, wherein the refractive index is 1.60 or less.
 3. A method of mounting a biological specimen on a substrate, comprising: depositing the biological specimen on the substrate; and contacting the biological specimen with the RIM solution of claim 1 to provide a mounted sample.
 4. The method of claim 3, further comprising drying the mounted sample, whereby the water-soluble polymer solidifies to provide a solidified polymer.
 5. The method of claim 4, wherein the water-soluble polymer solidifies in about 48 hours or less at room temperature.
 6. The method of claim 4, wherein the refractive index of the solidified polymer exceeds 1.47.
 7. (canceled)
 8. A solidified mountant, comprising a water-soluble polymer, wherein the polymer has a molar refraction (RLL) to molar volume (Vm) ratio of 0.27 to 0.34, and a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.318:1 to 4:1, and wherein the solidified mountant has a refractive index of 1.48 or greater.
 9. The solidified mountant of claim 8, wherein the weight ratio of the water-soluble polymer to the polyol is 0.5:1 or greater.
 10. (canceled)
 11. The RIM solution of claim 1, wherein the water-soluble polymer is uncharged.
 12. The RIM solution of claim 11, wherein the uncharged, water-soluble polymer is a poly(acrylamide), poly(methacrylamide), poly(methyl vinyl ether), poly(vinyl pyrrolidone) (PVP), polyvinyl alcohol (PVA), poly(2-ethyl-2-oxazoline), or poly(2-methyl-2-oxazoline).
 13. The RIM solution of claim 11, wherein the uncharged, water-soluble polymer comprises a monomer residue of N-R acrylamide or N-R methacrylamide, wherein R is methyl, ethyl, propyl, isopropyl or H; N,N-dimethylacrylamide, N,N-dimethylmethacrylamide, N,N-diethylacrylamide, N, N diethylmethacrylamide; N-2-hydroxypropyl methacrylamide); or a combination thereof.
 14. The RIM solution of claim 1, wherein the uncharged, water-soluble polymer is poly(N-methyl methacrylamide) (PMMAm).
 15. The RIM solution of claim 1, wherein the water-soluble polymer is charged.
 16. The RIM solution of claim 1, wherein the charged, water-soluble polymer is polyacrylic acid, polymethacrylic acid, poly(diallyldimethylammonium chloride), poly(sodium-4-styrenesulfonate), poly(ethyleneimine), poly(N,N-dimethylaminoethyl acrylate), poly(N,N-diethylethylamino acrylate), poly(allylamine), poly[bis(2-chloroethypether-co-1,3-bis[3-(dimethylamino)propyl]urea], or poly(vinylsulfonic acid, sodium salt).
 17. The RIM solution of claim 1, wherein the water-soluble polymer has a weight average molecular weight of about 1 kDa to about 100 kDa. 18.-20. (canceled)
 21. The solidified mountant of claim 8, wherein the refractive index is 1.48 to 1.54.
 22. The RIM solidified mountant of claim 8, wherein the refractive index of the solidified mountant matches the refractive index of soda-lime glass, borosilicate glass, or immersion oil. 23.-24. (canceled)
 25. The RIM solution of claim 1, further comprising an anti-oxidant. 26.-27. (canceled)
 28. A mounted biological specimen, comprising a biological specimen disposed on a substrate, wherein the biological specimen is embedded in the solidified mountant of claim
 8. 29. A kit for mounting a biological specimen on a substrate, comprising: a) the refractive index matching (RIM) solution of claim 1; and b) instructions for mounting the biological specimen on the substrate. 30.-32. (canceled) 